Neg 50 medium

Manufactured by Thermo Fisher Scientific

Sourced in United States

The NEG-50 medium is a laboratory equipment designed for tissue processing and embedding. It is a cryogenic medium used to prepare tissue samples for histological analysis and microscopic examination.

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Lab products found in correlation

Superfrost plus slide, by Thermo Fisher Scientific (2 mentions) Positively charged glass slides, by Avantor (1 mentions) Cryostat, by Leica (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Aperio versa 200, by Leica (1 mentions) Glutaraldehyde, by Clinisciences (1 mentions) Cm3050, by Leica (1 mentions) Colorfrost plus microscope slides, by Thermo Fisher Scientific (1 mentions) Axio observer z1 microscope, by Zeiss (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Vectashield, by Vector Laboratories (1 mentions)

Close spelling variants of this product

Neg-50TM Frozen Section Medium Richard-Allan Scientific™ Neg-50™ frozen section medium NEG-50 Tissue Tek medium Neg-50 Frozen Section Medium NEG-50 Neg-50TM

8 protocols using neg 50 medium

Preparing Dissected Fly Corpora Allata

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Whole flies with green fluorescent protein (GFP)–expressing corpora allata were briefly dipped in 75% ethanol to remove cuticle hydrocarbons before being fixed in 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS) with 0.3% Triton X-100 (PBST) for 60 min. After 3× 20-min washes with PBST, flies were transferred into Neg-50 medium (Thermo Fisher Scientific, 6502) and frozen in a dry ice/acetone cooling bath (−78°C). The frozen flies were sliced sagittally into 100-μm sections in a cryosection device and mounted directly on a glass slide with mounting medium (Neg-50). Confocal sections (four to seven sections at 3-μm intervals) were taken from the medial sections where corpora allata are seen (if not ablated).

Zhang S.X., Glantz E.H., Miner L.E., Rogulja D, & Crickmore M.A. (2021). Hormonal control of motivational circuitry orchestrates the transition to sexuality in Drosophila. Science Advances, 7(25), eabg6926.

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Tissue Fixation and Cryosectioning of Spinal Cord

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Rats were euthanized by transcardial perfusion. Rats were given a lethal dose of ketamine and dexmedetomidine (as above), and their thorax was opened to expose the heart. The perfusion cannula was inserted through the left ventricle into the aorta and a small incision was made in the right atrium. The rats were perfused with 300 mL phosphate-buffered saline (PBS) until the blood was cleared, followed by 400 mL of 4% paraformaldehyde (PFA) in PBS (0.1 M, pH 7.4) until tissue fixation was achieved. The spinal cord was dissected out, kept in 4% PFA for 24 h at 4 °C, and then transferred to 30% sucrose in PBS (0.1 M, pH 7.4) for at least 48 h. Then, a 12 mm long segment centered at the injury epicenter was cut out, embedded in NEG-50 medium (ThermoFisher Scientific, Waltham, MA, USA), and frozen at –20 °C for approximately 2 h. The tissue was longitudinally sectioned on a Leica cryostat at 20 µm thickness. Sections were mounted in series on positively charged glass slides (VWR, Radnor, PA, USA) and stored at –20 °C until further use.

Maldonado-Lasunción I., Haggerty A.E., Okuda A., Mihara T., de la Oliva N., Verhaagen J, & Oudega M. (2021). The Effect of Inflammatory Priming on the Therapeutic Potential of Mesenchymal Stromal Cells for Spinal Cord Repair. Cells, 10(6), 1316.

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Ex Vivo Evaluation of MK2i-Releasing Sheath

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For ex vivo study using MK2i-releasing PCL–HT sheath, the discarded human saphenous vein (HSV) was obtained from the patients undergoing peripheral or coronary vascular bypass surgery in accordance with approved IRB guidelines provided by Vanderbilt University Medical Center (VUMC). The HSV segment was cut into consecutive rings (2–3 mm in width), and PEG-based hydrogel was then filled inside HSV rings to prevent shrinkage. After that, the HSV rings were wrapped with each sample sheath (bare PCL or PCL–HT/Alexa 568–MK2i), and then they were incubated in RPMI 1640 medium containing 30% FBS, 1% L-glutamine and 1% PS under standard culture condition (37 °C and 5% CO₂). To visualize the delivered MK2i in the HSV tissue, Alexa 568 (red fluorescence) was conjugated to MK2i peptide before loading on the surface of PCL–HT sheath (3/3.5 mm in internal/external diameter and 3 mm in width). After ex vivo organ culture for 6 h and 3 days, the HSV tissues were frozen in NEG-50 medium (Thermo Scientific, USA), and sectioned (10 μm in thickness). The cross-sectioned tissues were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, USA), and then imaged using a fluorescent digital scanner (Aperio VERSA 200, Leica, Germany).

Lee Y., Le Thi P., Seon G.M., Ryu S.B., Brophy C.M., Kim Y., Park J.C., Park K.D., Cheung-Flynn J, & Sung H.J. (2017). Heparin-Functionalized Polymer Graft Surface Eluting MK2 Inhibitory Peptide to Improve Hemocompatibility and Anti-Neointimal Activity. Journal of controlled release : official journal of the Controlled Release Society, 266, 321-330.

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Tissue Preparation for Spinal Cord Imaging

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Mice were perfused with 4% w/v PFA (Polysciences, EM grade) and 0.1% v/v glutaraldehyde (GA; CliniSciences) in PBS pH 7.4. Perfused animals were kept on ice for 30 min, followed by the dissection of the brain and SC in PBS. Tissue was post-fixed in 4% w/v PFA in PBS ON at 4°C, rinsed in PBS, and kept in PBS containing 30% w/v sucrose ON at 4°C.
The SCs were cut into smaller segments of thoracic and lumbar regions and either used for the SC clearing protocol or embedded in Neg-50 medium (6502G, Thermo Scientific) before being frozen and stored at −80°C. A cryostat (CM3050S, Leica) with a chamber temperature of −23°C was used to cut sections of 4 µm nominal thickness. The sections were mounted in PBS on SuperFrost Plus slides (J1800AMNZ, Thermo Scientific), immunolabeled, covered with a glass coverslip (type #1.5, 0.17 µm thickness, catalog #6310134, VWR), and sealed with silicone polymer (picodent twinsil speed 22). Alternatively, slices were cut from freshly-frozen SC in Tissue-Tek, placed on glass coverslips, post-fixed with 2% PFA, labeled, and directly used for photoactivated localization microscopy (PALM) imaging in an open chamber in PBS.

Wiessler A.L., Hasenmüller A.S., Fuhl I., Mille C., Cortes Campo O., Reinhard N., Schenk J., Heinze K.G., Schaefer N., Specht C.G, & Villmann C. (2024). Role of the Glycine Receptor β Subunit in Synaptic Localization and Pathogenicity in Severe Startle Disease. The Journal of Neuroscience, 44(2), e0837232023.

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Brain Tissue Preparation for Cryosectioning

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Whole brains were dissected and fixed in 3% paraformaldehyde in PBS overnight at 4 °C with gentle shaking. Specimens were then cryoprotected with 30% sucrose in PBS before being embedded in NEG-50 medium (ThermoFisher). Sections (14 μm in thickness) were cut on a cryostat and collected on ColorFrost Plus microscope slides (ThermoFisher). Sections were dried on a slide warmer for 30 min prior to storage at −20 °C.

Ong T., Trivedi N., Wakefield R., Frase S, & Solecki D.J. (2020). Siah2 integrates mitogenic and extracellular matrix signals linking neuronal progenitor ciliogenesis with germinal zone occupancy. Nature Communications, 11, 5312.

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Cryopreservation and Imaging of Brain Sections

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Brains were fixed overnight in 4% PFA-PBS at 4°C, rinsed in PBS, and submerged in 30% sucrose-PBS until sinking (24 hours). Brains were frozen in NEG-50 medium (Richard-Allan Scientific) and cryostat sections (20 μm) were prepared and stored at −80°C until use. Sections were washed 3 times 10 minutes with PBS and incubated 1 μg/ml Hoechst 33342 (Invitrogen) for 30 minutes at room temperature. Sections were then mounted using Vectashield (Vector Laboratories) as mounting media. Images were acquired with a Zeiss Axio Observer Z.1 microscope coupled with an apotome2. Image measurements and quantifications were blindly performed using Fiji^{107 (link)}. Statistical significance was assigned by 2-way ANOVA in GraphPad Prism. We analyzed anatomically comparable regions from sections from 2 embryos from 2 IUEs (n=4) per injection construct.

Mangan R.J., Alsina F.C., Mosti F., Sotelo-Fonseca J.E., Snellings D.A., Au E.H., Carvalho J., Sathyan L., Johnson G.D., Reddy T.E., Silver D.L, & Lowe C.B. (2022). Adaptive sequence divergence forged new neurodevelopmental enhancers in humans. Cell, 185(24), 4587-4603.e23.

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Cerebral Organoid Sectioning and Staining

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Human cerebral organoids were generated from H9 human ES cells
(Wicell, WA09) as previously described (Lancaster and Knoblich,
2014)_ENREF_14. 64 days-old organoids were then embedded in Neg-50 Medium
(Richard-Allan Scientific) and sliced into 20 μm sections in a
cryostat. The slides were dried overnight before storing at
−20°C and stained as described below.

Tripathy R., Leca I., van Dijk T., Weiss J., van Bon B.W., Sergaki M.C., Gstrein T., Breuss M., Tian G., Bahi-Buisson N., Paciorkowski A.R., Pagnamenta A., Wenninger-Weinzier A., Martinez-Reza M.F., Landler L., Lise S., Taylor J.C., Terrone G., Vitiello G., Giudice E.D., Brunetti-Pierri N., D’Amico A., Reymond A., Voisin N., Bernstein J.A., Farrelly E., Kini U., Leonard T.A., Valence S., Burglen L., Armstrong L., Hiatt S.M., Cooper G.M., Aldinger K.A., Dobyns W.B., Mirzaa G., Pierson T.M., Baas F., Chelly J., Cowan N.J, & Keays D.A. (2018). Mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations. Neuron, 100(6), 1354-1368.e5.

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Ultrasound-guided Breast Cancer Biopsy

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Human sample acquisition and experimental procedures were carried out in compliance with regulations and protocols approved by the IRB-HSR at the University of Virginia in accordance with the U.S. Common Rule. In accordance with IRB Protocol #19272, breast cancer samples were collected as ultrasound-guided core needle biopsies during diagnostic visits from participants who provided informed consent. Each core biopsy was cut into two pieces, freshly cryoembedded in NEG-50 medium (Richard-Allan Scientific) in a dry iceisopentane bath, and stored at -80°C wrapped in aluminum foil. Cryosectioning and slide storage was performed exactly as described previously (13) .

Singh S., Sutcliffe M.D., Repich K., Atkins K.A., Harvey J.A., & Janes K.A. (2020). Pan-cancer Drivers are Recurrent Transcriptional Regulatory Heterogeneities in Early-stage Luminal Breast Cancer.

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